In this study of the role of microfloral beta-glucuronidase in colonic carcinogenesis, the effect of beta-glucuronidase inhibitor was evaluated. Starting at 5 weeks of age, male Donryu rats were fed either a semisynthetic diet or the same diet containing 0.1% beta-glucuronidase inhibitor as N-cyclohexyl-5-O-acetyl-2,4-O-p-methoxybenzylidene) -D-glucaro-1-amide-6,3-lactone (C-GAL). All animals were given s.c. injections of 7.4 mg azoxymethane (AOM) per kg body weight once a week for 11 weeks and followed for an additional 20 weeks. Most animals receiving the colonic carcinogen developed tumors in the colon, and a few also developed tumors in the small intestine. However, the number of tumors in the large intestine of the rats given C-GAL at the same time as AOM was significantly lower than in the control rats, especially in the proximal half of the colon, but those given C-GAL after AOM treatment had almost the same number of colon tumors as did the controls. It is concluded that, since bacterial beta-glucuronidase activity in the feces of rats given 0.1% C-GAL was significantly inhibited, intestinal microfloral beta-glucuronidase may play an important role in colonic carcinogenesis caused by AOM.